Kwon T K, Nordin A A
Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland 21224, USA.
Oncogene. 1998 Feb 12;16(6):755-62. doi: 10.1038/sj.onc.1201586.
A cdk2 binding domain on p27Kip1 located within the sequence of amino acids 53-85 was further characterized by generating a series of point mutations within amino acid residues 62-75. Two regions, FDF (residues 62-64) and GXY (residues 72 and 74), were identified within the beta hairpin region of p27Kip1. Mutations within these regions essentially completely inhibited the binding to in vitro translated cdk2 and cdk2/cyclin E complexes formed in vitro or in vivo. The p27Kip1 GST-fusion protein of the point mutation that replaces phenylalanine at residue 64 to alanine (F64A) showed approximately twofold less inhibition of cdk2 kinase activity. The cellular response to the introduction of the F64A mutant form of p27Kip1 was compared to that of p27Kip1 wild type by transfecting HeLa cells with constructs of full length sense and antisense coding sequences. Overexpression of the F64A mutant form of p27Kip1 bound significantly lower levels of cdk2 as compared to wild type and did not affect the cdk2 related kinase activity of the transfected HeLa cells. Overexpression of wild type p27Kip1 resulted in a reduction of the level of cdk2 kinase activity and effectively suppressed the growth of the transfected HeLa cells.
通过在氨基酸残基62 - 75内产生一系列点突变，对位于氨基酸序列53 - 85内的p27Kip1上的cdk2结合域进行了进一步表征。在p27Kip1的β发夹区域内鉴定出两个区域，FDF（残基62 - 64）和GXY（残基72和74）。这些区域内的突变基本上完全抑制了与体外翻译的cdk2以及在体外或体内形成的cdk2/细胞周期蛋白E复合物的结合。将残基64处的苯丙氨酸替换为丙氨酸（F64A）的点突变的p27Kip1 GST融合蛋白对cdk2激酶活性的抑制作用降低了约两倍。通过用全长有义链和反义编码序列构建体转染HeLa细胞，将引入p27Kip1的F64A突变体形式后的细胞反应与p27Kip1野生型的细胞反应进行了比较。与野生型相比，p27Kip1的F64A突变体形式的过表达结合的cdk2水平显著降低，并且不影响转染的HeLa细胞的cdk2相关激酶活性。野生型p27Kip1的过表达导致cdk2激酶活性水平降低，并有效抑制了转染的HeLa细胞的生长。
